The purpose of this study is to investigate intra-fraction setup variability in hypo-fractionated cranial and body radiotherapy; this is achieved by means of integrated infrared optical localization and stereoscopic kV X-ray imaging.We analyzed data coming from 87 patients treated with hypo-fractionated radiotherapy at cranial and extra-cranial sites. Patient setup was realized through the ExacTrac X-ray 6D system , consisting of 2 infrared TV cameras for external fiducial localization and X-ray imaging in double projection for image registration. Before irradiation, patients were pre-aligned relying on optical marker localization. Patient position was refined through the automatic matching of X-ray images to digitally reconstructed radiographs, providing 6 corrective parameters that were automatically applied using a robotic couch. Infrared patient localization and X-ray imaging were performed at the end of treatment, thus providing independent measures of intra-fraction motion.median ± quartile) 0.3 ± 0.3 mm, 0.6 ± 0.6 mm, 0.7 ± 0.6 mm for cranial, abdominal and lung patients, respectively. X-ray image registration estimated larger intra-fraction motion, equal to 0.9 ± 0.8 mm, 1.3 ± 1.2 mm, 1.8 ± 2.2 mm, correspondingly.According to optical measurements, the size of intra-fraction motion was (Optical tracking highlighted negligible intra-fraction motion at both cranial and extra-cranial sites. The larger motion detected by X-ray image registration showed significant inter-patient variability, in contrast to infrared optical tracking measurement. Infrared localization is put forward as the optimal strategy to monitor intra-fraction motion, featuring robustness, flexibility and less invasivity with respect to X-ray based techniques. Over the last few years, the development of Image Guided Radiation Therapy (IGRT) technologies has resulted in the design and realization of systems allowing precise patient setup and monitoring at each therapy fraction -3. The rWhen imaging techniques are used, the assessment of intra-fraction uncertainties in most cases is measured off-line at the end of irradiation. Actual real-time patient monitoring is usually achieved by tracking external surrogates, like Infra-Red (IR) markers ,12 or thet al. [In 2006 Linhout et al.  investigIn this work, we extend the analysis to frame-based and frameless hypo-fractionated (1-to-4 sessions) radiation therapy including cranial and extra-cranial treatment sites. An off-line analysis was performed on the log files storing the position of markers before and after treatment to measure 3D displacements. Stereoscopic X-ray images were acquired and matched before and after treatment to measure bony anatomy shifts. The specific aim of our study was the multimodal measurement of intra-fraction variations and the exploration of optimal strategies for monitoring the intra-fraction setup variability in high precision radiation therapy.We randomly selected 87 patients treated between May 2007 and March 2009 with hypo-fractionated stereotactic radiotherapy. The number of analyzed therapy sessions was 151 out the total of 231. Time limitations in the clinical routine and the absence of dedicated personnel on a regular basis did not allow us to acquire data at every fraction. Details about the patient population are presented in Table The treatment plan was calculated on a planning CT image set acquired with 3 mm slice thickness, using the BrainScan software . In cranial patients, isotropic margins ranging between 3 mm and 5 mm were added to the CTV  to define the PTV (Planned target volume). For extra-cranial treatments, anisotropic margins were defined on the basis of a breath hold CT scan acquisition around the target region, thus taking into account the tumor excursion from exhale to inhale . A slow CT scan was also acquired to ensure that tumor motion, during normal breathing, was included in the PTV. Additional 3 mm were added, in order to take into account setup uncertainties. The dose was normalized at the ICRU  reference point in order to obtain that the 95% of PTV was covered by the 95% isodose. The treatment was delivered with the support of a 3 mm multileaf collimator from Brainlab.et al. [The clinical protocol was designed and approved to monitor intra-fraction setup variability in selected patients. Head and neck patients . The user can manually exclude up to 70% of the image in order to remove ambiguous structures  from the registration process. The outcomes of image fusion are 6 corrective parameters that are applied through the robotic couch (ExacTrac Remote couch by Brainlab). A comprehensive technical description of the system can be found in Jin et al. [Patient setup was driven by the ExacTrac X-Ray system, an IGRT device featuring two sub-components; 1) an Infra-Red (IR) optoelectronic localizer and 2) a radiographic kV X-ray imaging device in double oblique projection. The IR localization features real time detection (30 Hz) of passive spherical markers (10 mm of diameter) with a ± 0.3 mm localization error. The field of view of kV images is 20.4 × 20.4 cmn et al. .At each therapy fraction, automatic patient alignment was perfomed by the optical system along the three linear directions . After that, two orthogonal kV images were acquired and automatically matched to DRR for computing setup corrections in 6 degrees of freedom  relying on bony anatomy. The correction was then performed through the 6 Dof robotic couch. A second X-ray acquisition was performed to measure the residual errors according to the imaging system. If residual translations and rotations were found below 1 mm and 1° respectively, the patient position was considered acceptable for treatment; otherwise the procedure was repeated iteratively to improve patient setup.PreIR) was acquired and averaged over at least 2 breathing cycles (8-10 seconds). The PreIR configuration represents the reference position for monitoring intra-fraction variations in our analysis, including the position of the target, which was automatically estimated by the ExacTrac software from the current arrangement of markers.Following patient setup procedures and before irradiation started, the 3D location of external markers (PostIR), that was averaged over the same time duration (8-10 seconds) that was used for PreIR. A post irradiation set of X-ray images was also acquired and registered to DRRs, for the estimation of post-irradiation 6 Dof roto-translation parameters (Ω) describing image-based intra-fraction motion. Off-line analysis of intra-fraction motion was expressed in terms of positional variations between pre and post irradiation and was performed following two approaches:In Figure PreIR and PostIR.1. Optical measurement: 3D displacements between 2. X-ray measurement: for consistency sake intra-fraction motion was quantified in terms of displacements of surface control points, accounting for information provided by pre-irradiation and post-irradiation image registration. This was achieved as follows:PreIR configuration, according to the residual corrective parameters provided by image matching before irradiation; this resulted in the PreIR* configuration of control points, accounting for residual patient setup errors as detected by X-ray imaging• Roto-translation of the PostIR configuration according to post-irradiation image registration (Ω correction vector), leading to PostXRay configuration.• Roto-translation of the PreIR* and PostXRay.• Calculation of 3D displacements between et al.[PostIR and PostXray configuration.A further analysis was performed on the target location. The center of mass of the tumor was estimated by applying the weighted strategy algorithm proposed by Riboldi et al.. The Euc-6) results for cranial, abdomen and lung patients are reported separately. In Figure th percentile) were 0.3 ± 0.3 mm - 1.0 mm, 0.6 ± 0.6 mm - 2.1 mm, 0.7 ± 0.6 mm - 1.4 mm  for optical measurements. Conversely, X-ray detected values measured 0.9 ± 0.8 mm - 2.9 mm, 1.3 ± 1.2 mm - 3.9 mm, 1.8 ± 2.2 mm - 7.1 mm.The normality test rejected the hypothesis of normal distribution in the population of 3D fiducial displacements. For this reason, data were analyzed following a non-parametric statistical approach. Due to statistically significant differences . As reported in Table The Wilcoxon matched pair test demonstrated statistical difference between optical and X-ray systems in each patient population (p < 10-3).Figure Figure One outlier, which is not displayed in the plots, showed 12.8 mm translation along the left-right direction, with acceptable values for the other directions (up to 2.2 mm translation in AP-direction and up to 0.6° yaw rotation). The optical system did not detect relevant shifts for this case.In this work, we measured intra-fraction motion in hypo-fractionated radiotherapy using a multimodal approach. Our main goal was to assess the quality of patient immobilization during treatment and to highlight the optimal measurement strategy . It is important to underline three relevant aspects of the implemented methodology:1. since extra-cranial treatments are performed in free breathing conditions, IR data were collected and averaged over at least two respiratory cycles to compensate possible respiration motion effects in a short time window. The effect of respiration movements was furthermore evaluated by measuring the standard deviation (std) of marker positions over each acquisition. The mean standard deviation ranged between 0.3 and 0.5 mm in the extra-cranial patient population. These values are due to the fact that most of the IR markers (4-5 over 7) were placed in correspondence of stable landmarks, like upper thorax or pelvis, thus leading to a robust measurement of patient position.et al. [2. The cranial patient population potentially represents an ideal situation, as intra-fraction motion is less relevant. However, the presence of the thermoplastic mask may represent a limitation because markers are typically placed onto the mask in our protocol. Therefore, the discrepancies found between the two measurements approaches can be due in part to movements of the patient within the mask, as suggested by Linthout et al. .3. The X-ray measurements were depurated from setup residuals, computed before treatment by means of image registration. This gave us more robustness in understanding and analyzing an X-ray based quantitative measurement of intra-fraction variations.et al., the main differences in our data analysis were the following:The analysis was performed off-line, by analyzing both the log files of markers position and the X-ray images stored immediately before and after irradiation. Compared to the methodology proposed by Linthout et al. the intra-fraction motion monitored by the optical localizer was evaluated in terms of the 6 Dof corrective parameters estimated by the Brainlab software. Here, we assessed the residual displacements on each external marker after optical measurements and then we estimated the isocenter position from the configuration of fiducials. This allowed us also to explore potential deformations in the configuration of markers, in order to test its reliability in patient setup control.1. In the work by Linthout et al. the comparison between the two sub-systems was performed by evaluating the corrective parameters coming from external point registration and image fusion. This kind of analysis has a conceptual flaw since an indeterminate number of roto-translations are able to match 2 different configurations in space at the same uncertainty level. Here, we roto-translated the external configuration of marker points according to image fusion and then we compared the 2 fiducial sets, point by point, to precisely examine the difference between the 2 approaches.2. In Linthout Measurements performed by the optical localizer showed on average sub-millimetric intra-fraction motion for both extra-cranial and cranial treatments. These results were confirmed when looking at target position, as estimated according to the external marker configuration under a rigid body assumption. Target position resulted essentially stable, with average intra-fraction motion within 1 mm. On the basis of these results, we can assume that immobilization devices and the automation of setup procedures help the patient to be comfortable and stable, thus leading to small intra-fraction variations.When comparing optical versus X-ray measurements, differences were on average 1-1.5 mm, with worst results in lung cases. It should be noted from Figures Patient setup verification should rely on multimodal monitoring systems  for the highest reliability in detecting and correcting geometric uncertainties. The reported analysis shows that optical tracking is able to provide robust measurement for the real-time detection of intra-fraction variations.AP: Antero-Posterior; ART: Adaptive Radiation Therapy; CBCT: Cone Beam Computed Tomography; CC: Cranio-Caudal; CT Computed Tomography; Dof; degrees of freedom; IGRT: Image Guided Radiation Therapy; IMRT: Intensity Modulated Radiation Therapy; IR: Infra-Red; kV: kilo Voltage; LR: Left-Right; MV: Mega Voltage; PostIR: 3D Marker position detected by the IR localizer after treatment; PostXRay: PostIR roto-translated according to the corrective parameters (Ω) estimated by image registration after treatment; PreIR: 3D Marker position detected by the IR localizer before treatment; PreIR*: PreIR roto-translated according to the verification parameters estimated by image registration before treatmentMFS had primary role in study design, data analysis, results interpretation and manuscript editing; BT and EP participated to data acquisition; MR and GB gave important contributions in data analysis, results interpretation, manuscript editing and final approval; CG was the medical physicist in charge of computing the dose and running the ExacTrac System; DA, GP were the physicians in charge of treatments; AP and RO gave final approval to conceptual study and manuscript.All authors read and approved the final manuscript.Authors declare that no competing interest existAuthors declare that written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


We further utilized UV radiation to etch m-SWCNTs from the networks, and a remarkable increase in the channel current on/off ratio (Ion/Ioff) from 11 to 5.6 × 103 was observed. Furthermore, we used the SWCNT-TFTs as gas sensors to detect methyl methylphosphonate, a stimulant of benchmark threats. It was found that the SWCNT-TFT sensors treated with UV radiation show a much higher sensitivity and faster response to the analytes than those without treatment with UV radiation.Single-walled carbon nanotube (SWCNT) random networks are easily fabricated on a wafer scale, which provides an attractive path to large-scale SWCNT-based thin-film transistor (TFT) manufacturing. However, the mixture of semiconducting SWCNTs and metallic SWCNTs (m-SWCNTs) in the networks significantly limits the TFT performance due to the m-SWCNTs dominating the charge transport. In this paper, we have achieved a uniform and high-density SWCNT network throughout a complete 3-in. Si/SiO It has and NH3[2. In thesand NH3[2,11.5 in the SWCNT transistors. Roberts et al. reported a self-sorting method to enrich and align the s-SWCNT on the surface of silicon and polymeric films by controlling the substrate surface chemistry [Several approaches have been developed for obtaining SWCNT-FETs with pure s-SWCNT in the channels such as selective synthesis of s-SWCNTs -18, posthemistry . It was 4. However, this technology requires pre-separating 95% enriched s-SWCNTs. Here, we reported on the deposition of random SWCNT networks from the SWCNT solution on the wafer and then radiated the nanotube networks utilizing UV to fabricate SWCNT-TFT sensors with high performance. A remarkable increase in the channel current on/off ratio from approximately 11 to 5.6 × 103 was observed after UV irradiation. We also used these SWCNT-TFTs to detect methyl methylphosphonate (DMMP), a stimulant of benchmark threats. The SWCNT-TFTs treated with UV radiation show a much higher sensitivity to the analytes than those without treatment with UV radiation.Random nanotube networks are easily deposited from the solution and represent an attractive path to large-scale device manufacturing -28. Howe2 wafer used is an n-doped Si (100) wafer with 300-nm oxide layer on the top of the Si wafer. DMMP and 3-aminopropyltrimethysilane (APS) were purchased from Sigma-Aldrich . Deionized water, ethanol, acetone, and toluene were used throughout all processes for cleaning. All reagents were analytical grade and were used without further purification.The carboxylic acid-functionalized SWCNTs were obtained from Carbon Solutions Inc. . The Si/SiO2 wafer substrate was ultrasonically rinsed with toluene, acetone, ethanol, and deionized water, followed by cleaning with a piranha solution (98% H2SO4:30% H2O2 = 3:1 (v/v)) for 2 h at 80°C. This clean wafer was immersed in APS aqueous solution (1.5 mM) for 2 h and then kept in a vacuum evaporator at 120°C for 1 h to form the amino-terminated monolayer on the surface of the Si/SiO2 substrate. The purified SWCNTs were ultrasonically dispersed in deionized water for 2 h. The pretreated Si/SiO2 substrate modified with an APS monolayer was immersed in the SWCNT suspension, followed by rinsing with ethanol and deionized water and drying with the aid of nitrogen flow. The morphology of the deposited SWCNTs was characterized by scanning electron microscopy .To fabricate our gas sensors, the Si/SiOThe SWCNT-TFT sensors were fabricated using the standard microfabrication procedures in our previous report . The int2 in a stainless steel. The output flow ratio of the diluted DMMP vapor was controlled by mass flow controllers. The different concentrations of DMMP were produced by regulating the flow ratio of the dilution gas to the flow rate of the carrier gas. The DMMP vapor was delivered into the sensing chip to test the sensor performance. Before testing, the sensor was cleaned by UV for about 2 min to remove the absorbed impurity from the SWCNT network  and then exposed to the DMMP.The homemade sensor testing system was established as in the previous reports . Nitroge2 blowing together with illumination with a lamp .The electrical signal of the sensor was monitored using a semiconductor parameter analyzer . After a stable baseline electrical signal was obtained, the DMMP vapor with the required concentration was introduced, and all sensing measurements were carried out at room temperature. The DMMP was desorbed from the SWNT surface by N2 wafer were fabricated with a solution-based self-assembly method [2 substrate was first functionalized with an amino-terminated monolayer. The carboxylic acid-functionalized SWCNTs de-bounded and dispersed in the water were easily absorbed and deposited on the amino-functionalized Si/SiO2 substrate via electrostatic interaction. Following nanotube deposition was the fabrication of SWCNT-TFTs. The source and drain electrodes were patterned by standard photolithography. The silicon substrate acts as a back gate, while SiO2 is used as the back gate dielectric. The schematic and SEM images of the SWCNT-TFT structure were shown in Figure The uniform and high-density SWCNT networks on the Si/SiOy method ,27. BrieIds)-source-drain voltage (Vds) curves of typical SWCNT-TFTs before and after UV radiation. The IdsVds curves were found to be very linear for Vds between −1 and 1 V, indicating the Ohmic contact between nanotubes and the electrodes -sIds versus Vg) of the sensor was measured using the Si substrate as a back gate. The measurement was operated at a constant source-drain voltage of 0.5 V and gate voltage between −20 and +20 V. As shown in Figure Vg) was swept from negative to positive values. The on/off ratio of the untreated SWCNT-TFTs is low (approximately 11) due to the contribution of the m-SWCNTs. After 5 to 20 min of light irradiation, the SWCNT-TFTs retained a p-type semiconductor behavior, but the on/off ratios increased from approximately 11 to approximately 5.6 × 103, suggesting that most of the m-SWCNTs were etched from the channels.The transfer characteristic  is defined as R = ΔG/G0 = (G − G0)/G0, where G0 and G are the conductance of nanotubes before and after exposure to the testing gas, respectively. Figure Vg = −10V) can enhance the sensitivity. As an electron donator, the adsorption of DMMP on the p-type SWCNTs is equivalent to applying a positive chemical gate voltage. From the IdsVg curve  convert m-SWCNTs to s-SWCNTs. A remarkable increase in the channel current on/off ratio from approximately 11 to 5.6 × 10The authors declare that they have no competing interests.TTC performed the experiment and measured the SEM, electronic, and gas response data. LMW designed the experiments and wrote the manuscript. DWS helped in the technical support for the experiments. YY participated in the measurements. JW and JZ provided useful suggestions. ZHZ and YW helped analyze the characterization results. YFZ supervised all of the study and provided financial support. All authors read and approved the final manuscript.


The high prevalence of HIV-1/AIDS in areas endemic for schistosomiasis and other helminthic infections has led to the hypothesis that parasites increase host susceptibility to immunodeficiency virus infection. We previously showed that rhesus macaques (RM) with active schistosomiasis were significantly more likely to become systemically infected after intrarectal (i.r.) exposure to an R5-tropic clade C simian-human immunodeficiency virus (SHIV-C) than were parasite-free controls. However, we could not address whether this was due to systemic or mucosal effects. If systemic immunoactivation resulted in increased susceptibility to SHIV-C acquisition, a similarly large difference in host susceptibility would be seen after intravenous (i.v.) SHIV-C challenge. Conversely, if increased host susceptibility was due to parasite-induced immunoactivation at the mucosal level, i.v. SHIV-C challenge would not result in significant differences between parasitized and parasite-free monkeys.Schistosoma mansoni; the other group was left parasite-free. Both groups were challenged i.v. with decreasing doses of SHIV-C. No statistically significant differences in 50% animal infectious doses (AID50) or peak viremia were seen between the two groups. These data strongly contrast the earlier i.r. SHIV-C challenge (using the same virus stock) in the presence/absence of parasites, where we noted a 17-fold difference in AID50 and one log higher peak viremia in parasitized monkeys (P<0.001 for both). The lack of significant differences after the i.v. challenge implies that the increased host susceptibility is predominantly due to parasite-mediated mucosal upregulation of virus replication and spread, rather than systemic effects.We enrolled two groups of RM and infected one group with The major impact of schistosome-induced increased host susceptibility is at the mucosal level. Given that >90% of all new HIV-1 infections worldwide are acquired through mucosal contact, parasitic infections that inflame mucosae may play an important role in the spread of HIV-1. Parasitic infections have been postulated to increase host susceptibility to HIV-1. We previously demonstrated that rhesus monkeys with active schistosomiasis were significantly more likely to become systemically infected after intrarectal exposure to an R5-tropic clade C simian-human immunodeficiency virus then were parasite-free control animals. However, we could not address whether parasites exert their effect at the mucosal level or systemically. To address the latter possibility, we measured the virus doses needed to achieve systemic infection after intravenous exposure of parasite-free or parasite-positive monkeys using the identical virus stock. None of the viral parameters tested in these two groups of monkeys were statistically significantly different. These results suggest that schistosomiasis modulates susceptibility to immunodeficiency virus acquisition predominantly at the mucosal level. Treatment for parasitic infections in populations at higher risk for HIV-1 acquisition could represent a cost-effective approach to slow the spread of HIV-1, which is predominantly transmitted through mucosal routes. However, we could not differentiate whether the observed effects of schistosome infection on SHIV-C acquisition were primarily the result of mucosal effects, such as inflammation and local reactions caused by the passage of parasite eggs, or were due to systemic effects.Using this R5 SHIV-C model, we tested the effect of 50 for the i.v. route (AID50-i.v.) by endpoint titration. The AID50-i.v differed only 3.3 fold between parasite-free and S. mansoni-positive RM, a difference that was not statistically significant. The magnitude of this difference was much smaller than that observed in the i.r. SHIV-C exposure study, suggesting that parasitic infections modulate host susceptibility to lentivirus acquisition predominantly at mucosal levels.To address the latter possibility, we intravenously (i.v.) challenged schistosome-free and schistosome-infected RM with the identical stock of SHIV-1157ipd3N4 as that used in the i.r. study env sequences of a primary HIV-1 clade C strain isolated from a recently infected Zambian infant. SHIV-1157ipd3N4 was engineered with an additional NF-kB site per long terminal repeat (LTR) in order to enhance viral replication. The methodology used to construct this virus and its biology are described elsewhere SHIV-1157ipd3N4 http://www.acmedsci.ac.uk/images/project/nhpdownl.pdf). CDC facilities are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Animal experiments were approved by the Institutional Animal Care and Use Committees at the CDC (IACUC ID: 1522) and the Dana-Farber Cancer Institute via a Collaborating Institution Animal Use Agreement. Because the experiments described here involved a virus that may cause an incurable disease, such as AIDS, discomfort, stress and pain may occur. Animals were closely monitored and observed for development of disease at least twice daily. If the animals are determined to be under stress or in discomfort, appropriate anesthetics and/or analgesics are administered as directed by the clinical veterinary staff. Euthanasia is also an option should treatment not alleviate stress. In the current study, no untoward clinical problems were noted, and none of the virus-infected monkeys progressed to AIDS. Animals were free of S. mansoni infection at the time the study was initiated.Chinese-origin adult female RM were housed at the animal facility of the Centers for Disease Control and Prevention (CDC) in Atlanta, GA. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the U.S. Public Health Services/National Institutes of Health, as well as according to the recommendations in the Weatherall report on “The Use of Non-human Primates in Research”  and percent eosinophils were calculated using standard methods. Hematology results, as well as CD4 and CD8 T-cell counts and ratios were determined by the Pathology Laboratory of the Yerkes National Primate Research Center . There was no evidence of fever, diarrhea, weight loss, or dysentery in these animals.Animals were anesthetized with ketamine and exposed percutaneously with 500 cercariae of a Puerto Rican strain of Anesthetized macaques were inoculated i.v. with 1 ml total volume of various dilutions of an identical SHIV-1157ipd3N4 stock that had been grown in RM PBMC. All animals were exposed within 1 hr after the viral stock was thawed.Peripheral blood samples were obtained by venipuncture and collected into Vacutainer cell-preparation tubes containing sodium citrate . Immediately after collection, plasma and PBMC were separated and quick-frozen. Plasma samples were stored at −80°C until viral RNA loads were assessed by real-time RT-PCR 50 values and statistical comparison of infectious doses were performed using the method of Spouge P-values are based on two-sided testing.Calculation of AID50-i.v. in control animals was 1 ml of a 1∶151,000 dilution of the viral stock   . This re50-i.v. in parasitized RM was 1 ml of a 1∶521,000 dilution of the viral stock compared to 1 ml of a 1∶151,000 dilution in parasite-free RM. This difference was also not statistically significant. Mean peak viral RNA loads in parasitized RM with systemic SHIV-C infection after i.v. challenge were not statistically different from those in RM without schistosomiasis  was 3.3 fold lower than that in non-parasitized controls   Fig. 2)Fig. 2), S. mansoni-infected RM are shown in 50-i.r. was 17-fold lower in monkeys with acute schistosomiasis compared to control RM. This difference was highly significant (P<0.001) The composite data for the i.r. and i.v. challenges using the same stock of SHIV-1157ipd3N4 in control and 50-i.r. was extrapolated to be 24.6 µl of the undiluted virus stock. In contrast, the AID50-i.v. was a mere 0.0034 µl (3.4 nl). We reasoned that these AID50 values are indirectly proportional to the relative risks of virus acquisition via a given route of exposure. Thus, we converted the AID50 values to 1/AID50 and normalized the value for the i.r. route to 1. The ratio of 1∶7,235 reflects the much greater susceptibility of parasite-free hosts to i.v. challenge compared to i.r. exposure. Interestingly, this ratio was much lower for S. mansoni-infected RM (7.6∶737 or 1∶42). This additional measure also reflects that S. mansoni-induced mucosal alterations preferentially increase host susceptibility to SHIV-C.Our data also allow a direct comparison of the virus doses needed to achieve systemic infection after i.v. versus i.r. SHIV-C challenge. In parasite-free RM, the AID50-i.v. for parasite-positive vs. parasite-free RM was 3.3-fold, which was not statistically significant; 2) in contrast, the difference in AID50-i.r. for parasite-positive vs. parasite-free RM was 17-fold (P<0.001) with very similar numbers of RM per experimental group; and 3) peak viral RNA loads differed only among i.r. challenged but not i.v. challenged groups of schistosome positive vs. parasite-free RM.Here we showed the results of end-point titrations of an identical SHIV-C stock as a function of the challenge route (i.v. versus i.r.) and the presence or absence of parasites in Chinese-origin RM. Together, our data showed: 1) the difference in AID50-i.v. was not statistically significant, whereas the difference for the AID50-i.r. was, needs to be interpreted with caution given the constraints of our study. Primate studies with their inherently high costs limit the numbers of experimental animals that can be enrolled. Much larger group sizes than we were able to use may find that parasitized hosts challenged intravenously develop systemic infection at statistically significantly lower viral inocula compared to parasite-free hosts. Our current data would predict that such a difference would be smaller than that for the mucosal virus challenge.The fact that the 3.3-fold difference in AIDS. mansoni infection.Overall, our data indicate that the increased host susceptibility to the R5 SHIV-C is predominantly due to parasite-induced changes at the mucosal level. We postulate that host factors of the mucosal innate and adaptive immune systems will be implicated. The nature of our virus challenge study mandated not disturbing the mucosa for tissue sampling. Future studies should seek to identify the exact mechanism(s) involved in mucosally-mediated upregulation of host susceptibility to an R5 AIDS virus in the presence of acute S. mansoni-infected monkeys to i.r. viral exposure may be related to the passage of eggs from the bloodstream, where the adult worms reside, to the lumen of the gut. Egg excretion in schistosome-infected hosts is dependent on cellular immune responses The increased susceptibility of S. haematobium infections, adult worms live in the blood vessels surrounding the bladder instead of the intestine. Eggs are excreted into the bladder and pass out of the host in the urine. However, the worms can reside in other areas of the urogenital vasculature and eggs often become lodged in other nearby organs, especially in women, and can cause female urogenital schistosomiasis (FUS). The latter is associated with “sandy patches” , neovascularization, and contact bleeding of the cervix and vaginal wall as a result of the inflammation in response to the eggs. In studies in Zimbabwe and Tanzania, FUS was associated with a 2.7 to 4-fold higher risk of HIV-1 acquisition In While our primate model studies have focused on the role of parasites in facilitating lentiviral acquisition, other investigators have examined the role of parasites on HIV-1 disease progression. Parasitic worms elevate viral loads and exacerbate destruction of CD4 cells in chronic AIDS virus infection. Both studies in experimental animals Helminth infections in pregnant women, with associated higher viral loads, may also increase the risk of mothers transmitting HIV-1 infections to their infants Our current study, together with previous reports, clearly suggests that schistosomiasis increases the risk of immunodeficiency virus acquisition predominantly at the mucosal level. While the conclusions of our earlier intrarectal challenge study are not directly applicable to other routes of HIV-1 exposure among humans, our primate model data provide a proof of concept that alterations in the mucosal environment enhance HIV-1 transmission in schistosome-infected hosts. Our conclusions are also consistent with studies showing FUS as a risk factor for HIV-1 infection and the greater role of heterosexual transmission of HIV-1 in sub-Saharan Africa than in more industrialized countries that do not have endemic schistosomiasis. Thus, treatment for parasitic infections in populations at high risk for HIV-1 acquisition could represent a cost-effective approach to slow the spread of HIV-1. Furthermore, treatment of parasites in individuals with HIV-1 coinfection could lower viral RNA loads, which in turn would decrease the infectivity of such persons and slow their HIV-1 disease progression.


Background. Alzheimer's disease (AD) is the most common cause of dementia in the elderly. AD is characterized by the accumulation of amyloid plaques and neurofibrillary tangles and by massive neuronal loss in the brain. There is epidemiologic and pathologic evidence that AD is associated with vascular risk factors and vascular diseases, contributing to cerebral hypoperfusion with consecutive stimulation of angiogenesis and upregulation of proangiogenic factors such as Angiopoietin-1 (Ang-1). Methods. In the present study, we measured Ang-1 serum levels in 42 patients with AD, 20 patients with mild cognitive impairment (MCI), and in 40 healthy elderly controls by ELISA. Results. We found significantly increased Ang-1 serum levels in patients with AD compared to control subjects (P = 0.003). There was no significant difference between MCI patients and healthy controls (P = 0.553) or between AD and MCI patients (P = 0.054). The degree of cognitive impairment as measured by the mini-mental status examination (MMSE) score was significantly correlated with the Ang-1 serum levels in all patients and healthy controls. Conclusions. We found significantly increased Ang-1 serum levels in AD patients. We could also show an association between Ang-1 serum levels and the cognitive status in all patients and healthy controls. Thus, serum Ang-1 could be a potential candidate for a biomarker panel for AD diagnosis. Epidemifunction –14.Several growth factors or their lack have been implicated in the pathogenesis of AD. Angiopoietins are a family of growth factors specific for the vascular endothelium . The speThe current study aimed to examine Ang-1 serum levels in AD patients, MCI patients, and healthy elderly controls and to examine the association with the degree of cognitive impairment as measured by the mini-mental state examination (MMSE).A total of 42 patients with AD, 20 patients with MCI and 40 healthy elderly controls, were included in the study. Baseline characteristics and demographic parameters are displayed in Peripheral venous blood was sampled into serum tubes between 08:00 and 09:00 hours (fasting state) in order to take in account a possible circadian rhythm. Tubes were immediately immersed in melting ice. To minimize the source of platelets, serum was centrifuged within 30 min after sampling and stored at −20°C until further analysis.Serum levels of Ang-1 were measured using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's  instructions.P < 0.05. We conducted a bivariate correlation analysis (Pearson correlation) between age, MMSE scores, and Ang-1 serum levels. The two-tailed t-test was used to assess differences between two groups in case of normal distribution. The Mann-Whitney U-test was used to assess differences between two groups in case of nonnormal distribution. The chi-square test was used to assess differences in gender between two groups.All statistical analyses were carried out using the statistical analysis software package SPSS 19 . For comparisons of Ang-1 serum levels between subject groups , we calculated a univariate ANOVA. The data are presented as mean ± S.D. Significance for the results was set at P = 0.003). Pairwise comparisons with an independent t-test revealed a significant difference between AD patients and controls -0.596; P = 0.553) nor between AD and MCI patients (t(60) = 1.969; P = 0.054).Accounting for the age difference between controls and AD patients, we included age as a covariate for the ANOVA. We found a difference in Ang-1 serum levels between the three different groups (n = 102) .There is a significant positive correlation between the MMSE score (as a measure for cognitive status) and Ang-1 serum levels seen in all patients and healthy controls (The major findings of the present study are as follows. (1) Ang-1 serum levels are significantly higher in AD patients compared to healthy controls. (2) Taking into account the confounding effect of altered Ang-1 serum levels and MMSE scores in AD patients, Ang-1 serum levels are significantly inversely correlated with MMSE as a measure for cognitive status in the whole study population. This indicates that a lower degree of cognitive functioning is associated with higher Ang1-serum levels.Hypoperfusion of the brain caused by atherosclerotic changes of the vessels is assumed to play an important role in the pathogenesis of AD . This hyAngiogenesis is a complex process and consists of several discrete steps beginning with endothelial activation. Under normal conditions, endothelial activation is reversible and self-limiting. In AD, there is a continuous vascular activation induced by hypoperfusion, and factors and processes associated with angiogenesis can be found in the brain . Howeverβ deposits and neurofibrillary tangles in the brain from AD patients. There is evidence for inflammatory toxicity in the AD brain. For example, complement fixation and lysis of neurites can be demonstrated ultrastructurally [Besides angiogenesis, inflammation plays another important role in the pathogenesis of AD . Neverthcturally , 33. Sevcturally . Under icturally , 35. In cturally . Thus, tIn conclusion, we found significantly increased Ang-1 serum levels in AD patients. We could also show an association between Ang-1 serum levels and the cognitive status in all patients and healthy controls. Thus, serum Ang-1 could be a potential candidate for a biomarker panel for AD diagnosis.


Cholangiocarcinoma (CC) is a highly malignant carcinoma. We attempted to clarify the prognostic significance of c-Met overexpression and its association with clinicopathological factors in patients with CC.high and c-Metlow groups. Clinicopathological factors and outcomes were compared between the groups. c-Met and epidermal growth factor receptor (EGFR) expression was also examined in 10 CC cell lines.One hundred and eleven patients with intrahepatic CC (IHCC) and 136 patients with extrahepatic CC (EHCC) who had undergone curative surgery were divided immunohistologically into c-Methigh expression was demonstrated in 11.7% of IHCC and 16.2% of EHCC. c-Methigh expression was significantly correlated with the 5-year survival rate for CC overall (P=0.0046) and for IHCC (P=0.0013), histopathological classification in EHCC, and for EGFR overexpression in both IHCC and EHCC. Coexpression and coactivation of c-Met and EGFR were also observed in CC cell lines. Multivariate analysis revealed that c-Methigh expression was an independent predictor of poor overall and disease-free survival in patients with IHCC.The positivity of c-Met was 45.0% in IHCC and 68.4% in EHCC; c-Metc-Met overexpression is associated with EGFR expression and is a poor prognostic factor in CC. Cholangiocarcinoma (CC) is a highly malignant invasive carcinoma arising through malignant transformation of cholangiocytes. Epidemiologic studies have demonstrated that the incidence and mortality rates of this disease, especially those of intrahepatic CC (IHCC), are increasing worldwide , such as epidermal growth factor receptor (EGFR), vascular epithelial growth factor (VEGF) and c-Met, are promising targets for treatment of CC .α6β4, CD44, plexin B, Fas and other RTKs such as RON, EGFR and ErbB2 . Activation of HGF-c-Met signalling initiates cell invasiveness and triggers metastasis through direct involvement of tumour angiogenesis , in the largest cohort (111 cases of IHCC and 136 cases of extrahepatic CC (EHCC)) of surgical specimens of CC. We also examined the expression of c-Met and EGFR in CC cell lines.A total of 247 patients with CC were examined in the present study. The patients had undergone surgery and been diagnosed histologically as having adenocarcinoma of the bile duct, except for cancer of gallbladder and ampulla of Vater, at the National Cancer Center Hospital, Tokyo, between February 1990 and July 2005. Patients who had other malignancies or had died within four weeks after surgery were excluded. Clinical and pathological data were obtained from the medical records of the patients. To examine the correlations of c-Met with other RTKs , qualified cases including previous data for overexpression of these molecules (The studied patients included 168 men and 79 women ranging in age from 33 to 82 years (median 65 years), who had been observed for periods ranging from 1.4 to 204.5 months (median 29.8 months). The cases were divided into two groups, IHCC and EHCC, in accordance with the TNM Classification of Malignant Tumours  defined μm thick) cut from representative paraffin-embedded serial tissue slices were prepared on silicone-coated slides for IHC evaluation. Sections cut through the maximum tumour diameter were selected for IHC evaluation. The sections were deparaffinised in xylene, and rehydrated through graded concentrations of ethanol (50–100%). Endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide solution for 30 min. The antigens were retrieved by heating in a pressure cooker at 121°C for 10 min in 0.01 M citrate buffer. The tissue sections were incubated overnight at 4°C with anti-c-Met primary antibody  at a dilution of 1 : 50. After a washing in PBS, the sections were treated with Envison+ Dual link reagent at room temperature for 30 min. 3,3′-Diaminobenzidine tetrahydrochloride was used as the chromogen, and the tissue sections were counterstained with haematoxylin.Immunohistochemistry (IHC) was performed on 247 formalin-fixed, paraffin-embedded tissue sections. Immunohistochemical staining for c-Met was performed using a polymer-based method ). Serial sections (4 low (0 or 1+) or c-Methigh (2+), for purposes of statistical analysis. The sections were evaluated by three observers, MM, HO, and TS, without knowledge of the clinical data. HO and TS are board-certified pathologists. IHC of EGFR and assessment of its expression were done as described previously , 1% Triton X-100, and 150 mM NaCl with a complete protease inhibitor cocktail  and a phosphate inhibitor cocktail . The protein concentration was determined using a Bio-Rad protein assay kit . Lysates (5 μg protein per well) were separated by SDS-PAGE, then transferred to polyvinylidene difluoride membranes . The membranes were blocked with 5% skim milk in PBS for 30 min and then probed with the following primary antibodies: anti-c-Met , anti-phospho-Met , anti-EGFR , and anti-phospho EGFR  at 4°C overnight. After washing with PBS-Tween 20 (0.5%), the membranes were re-blocked and then incubated at room temperature for 1 h with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibody at a dilution of 1 : 1000. Following three washes, bands were visualised using the ECL Western Blotting Detection Reagents . Anti-β-actin  was used as a loading control.Subconfluent cells were lysed at 4°C for 30 min using lysis buffer containing 10 mχ2-test. Cumulative survival rates and survival curves were calculated by the Kaplan–Meier method, and log-rank test was performed for the comparison of survival curves between low and high groups defied by c-Met expression level. The Cox proportional hazards model was used to estimate the hazard ratio and 95% confidence interval of each outcome (tumour death and recurrence). Multivariate analysis was performed for factors selected as risk factors by univariate analysis, except for UICC pT and UICC stage, which are composed of other factors. Correlations between the intensity of c-Met and that of EGFR in IHC or Western blotting were determined by Spearman's rank correlation. Statistical analysis was done using the Statview 5.0 statistical software package . The level of significance was set at P<0.05.Correlations between the results of IHC and clinicopathological factors were determined by Fisher's exact probability test, except for histopathological classification, which was analysed by c-Met staining was localised in both the cell membrane and cytoplasm of CC cells . Strong Expression of c-Met, phospho-Met, EGFR, and phospho-EGFR in ten CC cells and one gastric cancer cells were estimated by Western blotting . ExpressP=0.0063), and histopathological classification (P=0.0239) and overexpression of EGFR (P=0.0056) in EHCC. No other clinical factors were associated with c-Met expression.The relationships between c-Met expression and clinicopathological factors of IHCC and EHCC were evaluated and are shown in high and c-Metlow groups was 15.4 and 41.1% (P=0.0013) for IHCC and 40.9 and 45.8% (P=0.1396) for EHCC, respectively (Five-year survival for patients in the c-Metectively . We thenectively .high group tended to have a poor 5-year survival rate, but not to a significant degree. Univariate analysis also showed that c-Methigh was not a significant factor for survival. Therefore, multivariate analysis was not performed for EHCC.In EHCC, the c-MetIn the present study, we have demonstrated the importance of c-Met overexpression in the prognosis and treatment of CC. We found that c-Met expression was correlated with EGFR overexpression in CC, and that it was also a significant prognostic factor in IHCC. In previous studies, the frequency of c-Met overexpression ranged from 21 to 58% in IHCC (Simultaneous expression of c-Met and EGFR has been observed in clinical specimens of primary chordoma (Collectively, it seems reasonable that efficient molecular therapy for CC should target multiple kinases such as c-Met, EGFR, and VEGFR. c-Met activation is regarded as one of the molecular mechanisms involved in the acquisition of resistance to anti-EGFR therapy, as activation of the alternative RTK pathway would bypass the EGFR pathway . TherefoIn conclusion, c-Met overexpression is significantly correlated with overexpression of EGFR in CC and with prognosis in IHCC. Further molecular investigation of the interaction between EGFR and c-Met in this fatal disease is urgently needed.


In the current study, microRNA (miRNA) microarrays were used to detect differentially expressed miRNAs in the myocardial tissues of rat models under stress, to screen target miRNA candidates for miRNA therapy of stress-induced myocardial injury. Rats were bound and suspended in order to induce acute stress (AS) and chronic stress (CS) models. miRNA microarrays were used to detect differentially expressed miRNA in the myocardial tissues of the stressed and control groups. In comparison to the normal control, there were 68 differentially expressed miRNAs in the AS model, of which 32 were upregulated and 36 were downregulated. There were 55 differentially expressed miRNAs in the CS model, of which 20 were upregulated and 35 were downregulated. Of the 123 miRNAs, 15 miRNAs were differentially expressed between the AS and CS groups, of which four were significantly upregulated  and 11 were downregulated . The stress of being bound and suspended induces myocardial injury in the rats. Myocardial injury may cause the differential expression of certain miRNAs. Psychological stress may lead to the significant upregulation of rno-miR-296, rno-miR-141, rno-miR-382 and rno-miR-219-5p in addition to the significant downregulation of miR-135a and miR-466b. Stress is a non-specific systemic adaptive response of the body stimulated by a variety of internal and external environmental, social and psychological factors, also known as the stress response. Sympathetic and parasympathetic nervous system balance and hypothalamic-pituitary-adrenal (HPA) axis function alter under stress stimulation . FurthermicroRNA (miRNA) is small non-coding RNA of 18–26 bp. miRNA causes the degradation of, or inhibits the translation of, target mRNA by pairing with specific bases of the target mRNA and is thus involved in the post-transcriptional regulation of gene expression ,5. StudiIn the present study, rat models of myocardial injury induced by psychological stress were established and miRNA microarrays were used to analyze the changes in the miRNA expression profiles of injured myocardial tissues. Furthermore, the role of miRNA in stress-induced myocardial injury was studied and an attempt was made to locate the target miRNAs which regulate stress-induced myocardial injury, which may provide the basis for the development of new drugs for the prevention and treatment of stress-induced myocardial injury.A total of 24 eight-week-old male Wistar rats with body weights of 220±10 g were supplied by the Experimental Animal Center of Jilin University Norman Bethune Medical Division . The laboratory was disinfected, quiet and well-ventilated. The animals were provided with free access to food and water and were housed at a temperature of 22±2°C, with 50% humidity and a 12 h natural light-dark cycle. The rats were randomly divided into the normal control group , the chronic stress group  and the acute stress group . The Jilin University Animal Ethics Committee approved the protocol for this study (no. 2012036).The method for modeling stress in a rat has been described previously . For CS Statistical processing for this section was performed as follows: the SPSS 16.0 software package  was used for statistical analysis and measurement data were stated as the mean ± standard deviation. Comparisons were performed using the mean adopted t-test, and P<0.01 was considered to indicate a statistically significant difference.The rats underwent thoracotomies under anesthesia in order to remove their hearts. The hearts were washed using 0.2 mol/l phosphate buffer saline (pH 7.0) and stored in liquid nitrogen. Next, ~100 mg myocardial tissues were frozen and crushed using a BioPulverizer™ , mixed with 1 ml TRIzol reagent , and homogenized. Total RNA was extracted using the TRIzol reagent method. Extracted RNA was dissolved in RNAse-free water and incubated at 60°C for 10 min.260/A280 and A260/A230 ratios were calculated to determine the purity of RNA. The RNA concentration was calculated according to the formula: A260 × 40 ng/μl. Denaturing agarose gel electrophoresis was performed. Briefly, 3 μg RNA were samples stored in RNAse-free water mixed with formaldehyde-containing loading buffer (containing 10 μg/ml ethidium bromide) at a volume ratio of 1:3 and immediately incubated at 70°C for 15 min in order to denature the samples. Electrophoresis was performed at a voltage of 6 V/cm for 10–15 min, and gel images were captured using a gel image processing system .Absorbance at 260, 280 and 230 nm was measured using an ultraviolet spectrophotometer . A2O) was incubated at 37°C for 30 min and then at 95°C for 1 min to terminate the reaction, and immediately placed in an ice bath for 10 min. Following mild centrifugation , 3 μl labeling buffer, 1.5 μl fluorescent labels (Hy3™ for the stress group or Hy5™ for the control), 2 μl DMSO and 2 μl labeling enzyme were sequentially added in an ice bath. The system was incubated at 16°C for 1 h and subsequently at 65°C for 15 min in order to terminate the labeling reaction, and stored at 4°C following mild centrifugation.miRNA was labeled using the miRCURY™ Array Power Labeling kit  in accordance with the manufacturer’s instructions. Briefly, 4 μl calf intestine phosphatase (CIP) reaction solution  were hybridized in accordance with the manufacturer’s instructions. Briefly, 180 μl reaction mixture  was incubated at 95°C in the dark for 2 min and subsequently placed in an ice bath for 10 min. At the same time, the miRCURY Array chip was assembled according to the manufacturer’s instructions . The reaction mixture was loaded through the loading port and the 1X hybridization buffer was used to fill the hybridization chamber. The microarray chip was packaged in a protective bag and placed vertically into water (95°C) for 12 h, followed by drying in an oven at 56°C over night. The chip was washed using the miRCURY Array Wash Buffer kit  according to the manufacturer’s instructions, followed by centrifugation at 200 × g for 5 min to dry the chip. The scanning procedure was performed using an Axon GenePix 4000B microarray scanner  to obtain the scanning profiles, followed by data analysis and analysis of the significant difference using GenePix Pro6.0 software .An experienced operator utilized the GenePix Pro6.0 software for data processing and analysis. The signal points included in the subsequent analysis should conform to two prerequisites: i) The signal intensity of the red and green channels is >0; and ii) the signal to noise ratio (SNR) of the two channels is >1, or either SNR is >2. The weak signal points that did not conform to the two prerequisites were excluded from the subsequent analysis. miRNA was also excluded provided it contained 3 or 4 weak signal points in a total of four repeat points.Lowess standardization (intra-chip standardization) was performed to remove dye intensity-dependent deviation. Scale standardization (inter-chip standardization) was used to reduce the experimental or sample errors between the different chips. Four repeat points in the chip were merged by calculating the median of the Hy5/Hy3 ratio of each point. Subsequently the differentially expressed miRNAs were determined using the statistical Student t-test. P<0.05 was considered to indicate a statistically significant difference. Fold change (stress/control) ≥1.5 indicated upregulation and <0.67 indicated downregulation.Rats in the AS group appeared to be in a stressed state immediately following the surgery, but in the CS group, stress appeared after five days. The HPA system excites the stress stimulus and may be involved in abnormal secretion of corticotropin-releasing hormone (CRH), ACTH and three glucocorticoid hormones. The paraventricular nucleus of the hypothalamus secretes CRH under stress. The adenohypophysis CRH receptors perceive the change in ACTH and cause the adrenal cortex to secrete ACTH. ACTH is a key hormone of the HPA axis. Therefore, ACTH was selected to evaluate the stress state. As shown in 260/A280 ratio of RNA solution is a method for detecting RNA purity and values close to 2.0 are considered to represent pure RNA. A ratio <1.8 indicates sample contamination. A ratio >2.0 indicates RNA hydrolysis. The ratio range between 1.8 and 2.1 is acceptable. In addition, the A260/A230 ratio should be >1.8 for pure RNA. As demonstrated in The AAccording to data processing and analysis, specific miRNAs were identified to be differentially expressed in the stress model by comparing them with the normal control group  and III.Excessive psychological stress leads to myocardial injury by changing the function of the sympathetic nervous system and the HPA axis. Studies have demonstrated that miRNA expression is tissue-specific and is involved in the formation and maintenance of tissue specificity during biological development –18. In mThe present study analyzed miRNA expression profiles in the myocardial tissues of AS and CS rat models using miRNA microarray technology and identified specific differentially expressed miRNAs. In the differentially expressed miRNAs of the AS and CS models, 15 miRNAs were differentially expressed in the AS and CS models. Of these 15 miRNAs, rno-miR-296, rno-miR-141, rno-miR-382 and rno-miR-219-5p were significantly upregulated, particularly miR-296 , and 11 miR-125b, miR-146, miR-150, miR-199a, miR-21, miR-129, miR-341 and miR-451 have been confirmed to play an important role in the different developmental stages of the cardiovascular system . They aret al (In two stress-induced models, rno-miR-296 was the most significantly upregulated and miR-135a was the most significantly downregulated. Würdinger et al  observedet al . In the et al (in vitro translation of NR3C2 to regulate the angiotensin-aldosterone system balance. In the present study, miR-135a was significantly downregulated. We hypothesized that miR-135a may interact with the target genes to inhibit sympathetic nerve excitation and suppress the HPA axis and the renin-vascular angiotensin system, resulting in the release of a variety of stress hormones, including catecholamines, cortical hormone, pancreatic glucagon and renin, thus protecting the myocardium from injury.NR3C2 is a ligand-dependent transcription factor associated with steroid hormones. The transcription factor regulates the balance of water and ions and affects blood pressure by regulating water-sodium retention. Sõber et al  verifiedin vitro to explore the relationship between the miRNA and target genes in cardiomyocytes, which the authors believe should be studied further. The specific miRNAs found in the present study are the key to the further study of miRNA function.In conclusion, rno-miR-296, rno-miR-141, rno-miR-382, rno-miR-219-5p, miR-135a and miR-466b may be involved in stress at the molecular level, thus causing myocardial injury. The development of stress-induced myocardial injury is a complex biological process and involves a variety of mechanisms. After cells receive external stimuli, stimulatory signals are transferred through multiple pathways or channels into the cells, causing a series of reactions. Altering the conditions in these cells may lead to the activation of the cell death pathway, particularly the activation of the mitochondrial death mechanism, causing a death cascade reaction, which includes cell necrosis and apoptosis. miRNA may cause degradation of the target mRNA or inhibit its translation by pairing with the specific base of target mRNA and thus play a role in post-transcriptional regulation. Multiple miRNAs can jointly regulate the same target gene, and multiple target genes are capable of interacting with the same miRNA. By regulating the level of mRNA transcription, miRNA controls the amount of protein synthesis, thus regulating the occurrence and development of cardiovascular diseases. Once the specific miRNA is screened out, the target gene may be predicted. Furthermore, cardiomyocytes of miRNA inhibition and overexpression, following gene transfer using the miRNA mimic and miRNA inhibitors methods, may be cultured


Dyadobacter fermentans  is the type species of the genus Dyadobacter. It is of phylogenetic interest because of its location in the Cytophagaceae, a very diverse family within the order ‘Sphingobacteriales’. D. fermentans has a mainly respiratory metabolism, stains Gram-negative, is non-motile and oxidase and catalase positive. It is characterized by the production of cell filaments in aging cultures, a flexirubin-like pigment and its ability to ferment glucose, which is almost unique in the aerobically living members of this taxonomically difficult family. Here we describe the features of this organism, together with the complete genome sequence, and its annotation. This is the first complete genome sequence of the sphingobacterial genus Dyadobacter, and this 6,967,790 bp long single replicon genome with its 5804 protein-coding and 50 RNA genes is part of the  Genomic  Encyclopedia of  Bacteria and  Archaea  project. T (= DSM 18053 = ATCC 700827 = CIP 107007) is the type strain of Dyadobacter fermentans, which is the type species of the genus Dyadobacter. D. fermentans was described by Chelius and Triplett in 2000 [Dyadobacter  was co-isolated from the same material, although no microscopic evidence has been presented to date that members of these species were living within the plant tissue [Strain NS114D. fermentans were regularly found in cysts of the soybean nematode Heterodera glycines [D. fermentans was also isolated from contaminated soil as a result of its ability to grow on 7,8-benzoquinoline [D. fermentans, a fact suggesting that members of this taxon are not very abundant: three clones of uncultured bacteria of a deep sea sediment collected at the Western Tropical Pacific Warm Pool in the Pacific Ocean  and one of the activated sludge of a membrane bioreactor (EU283373) are recorded (as of May 2009). Intrageneric similarity within the genus Dyadobacter is rather low [Members of the species glycines . D. fermuinoline , a nitrother low .D. fermentans has been recognized by its flexirubin-like yellow pigment and by its growth as flocculent filaments of ovoid rods in old cultures [T occur polarly attached in groups of 2-4 cells during logarithmic growth, cells being frequently arranged at an angle to give V-formations. As the culture ages, irregular filaments or ovoid rods form . The slight differences between the genome data and the reported 16S rRNA gene sequence are probably the result of sequencing errors in the previously reported sequence data.T is dominated by unsaturated, and saturated iso-branched, straight chain unsaturated and large amounts of iso-branched, hydroxylated species. Major components are iso-C15:0-2OH and/or C16:1ω7c (43.5%), C16:1 ω5c (17.5%) and iso-C15:0 (16.8). Considerable amounts of the 3-hydroxylated fatty acids iso-C15:0-3OH, C16:0-3OH and iso-C17:0 -3OH are detected [T. The main component reported for the closely related type strains of D. ginsengisoli and D. alkalitolerans is menaquinone MK-7 [T contain spermidine as the major cellular polyamine and putrescine, cadaverine and spermine as minor components. The latter compound was not detected in any of the three other strains studied to date of the family Cytophagaceae, representing Flexibacter flexilis, Microscilla marina, and D. beijingensis [Dyadobacter.The whole cell fatty acid pattern of strain NS114one MK-7 . Cells oingensis . The pol Genomic  Encyclopedia of  Bacteria and  Archaea  project. The genome project is deposited in the Genomes OnLine Database [This organism was selected for sequencing on the basis of its phylogenetic position, and is part of the Database  and the D. fermentans NS114T, DSM18053, was grown in DSMZ medium 830 (R2A Medium) at 28°C [et al. [ at 28°C . DNA was [et al. .http://www.jgi.doe.gov). Draft assemblies were based on 86,260 total reads. The Phred/Phrap/Consed software package (http://www.phrap.com) was used for sequence assembly and quality assessment [The genome was sequenced using a combination of 8 kb and fosmid DNA libraries. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website (sessment -24. Aftesessment . Gaps beGenes were identified using Prodigal  as part The genome is 6,967,790 bp long and comprises one main circular chromosome with a 51.4% GC content . Of the


In this paper, single-stranded DNA (ss-DNA) is demonstrated to functionalize graphene (GR) and to further guide the growth of PtAu bimetallic nanoparticles (PtAuNPs) on GR with high densities and dispersion. The obtained nanocomposites (PtAuNPs/ss-DNA/GR) were characterized by transmission electron microscopy (TEM), energy-dispersive X-ray spectrometer (EDS), and electrochemical techniques. Then, an enzyme nanoassembly was prepared by self-assembling glucose oxidase (GOD) on PtAuNP/ss-DNA/GR nanocomposites (GOD/PtAuNPs/ss-DNA/GR). The nanocomposites provided a suitable microenvironment for GOD to retain its biological activity. The direct and reversible electron transfer process between the active site of GOD and the modified electrode was realized without any extra electron mediator. Thus, the prepared GOD/PtAuNP/ss-DNA/GR electrode was proposed as a biosensor for the quantification of glucose. The effects of pH, applied potential, and temperature on the performance of the biosensor were discussed in detail and were optimized. Under optimal conditions, the biosensor showed a linearity with glucose concentration in the range of 1.0 to 1,800 μM with a detection limit of 0.3 μM (S/N = 3). The results demonstrate that the developed approach provides a promising strategy to improve the sensitivity and enzyme activity of electrochemical biosensors. It is well known that the diabetes mellitus is one of the leading causes of death and disability in the world which can be easily diagnosed and managed by the determination of blood glucose . The gre2-hybridized carbon structure in a single-atom-thick sheet, has rapidly emerged as one of the most attractive materials 3-/4- and the GC electrode. After GOD assembled on the PtAuNP/ss-DNA/GR electrode (curve d), the semicircle dramatically increased, indicating that the presence of the GOD molecules on the electrode surface blocked the electron transfer.In electrochemical impedance spectroscopy measurements, the semicircle diameter of impedance equals the electron transfer resistance (Ret), which controls the electron transfer kinetics of the redox probe at the electrode interface and is an important parameter. Figure 2-saturated PBS (curve a), O2-saturated PBS without 1.0 mM glucose (curve b), and O2-saturated PBS containing 1.0 mM glucose (curve c). As shown in Figure 2-saturated solution was observed, indicating that GOD undergoes a reversible electrochemical reaction. The shape of redox peaks for the direct electron transfer of GOD dramatically changed in the presence of O2  represent the oxidized and reduced form of GOD, respectively.where GOD (FAD) and GOD , dopamine (1.0 mM), and uric acid (1.0 mM) on the amperometric response of 1 mM glucose, and the response is shown in Table .0 mM, doThe reproducibility and repeatability of the developed biosensor were determined. In a series of 10 biosensors prepared in the same way, a relative standard deviation (RSD) of 5.1% was obtained toward 0.1 mM glucose, indicating the reliability of the method. A set of 10 different amperometric measurements for 0.1 mM glucose with a single sensor yielded an RSD of 4.6%.The stability of the glucose biosensor was explored. The proposed biosensor was stored at 4°C in the refrigerator. The response to 0.1 mM glucose was tested each week; after 21 days of storage, the response of the biosensor only had a decrease of 5.5% compared to the initial response, which shows long-term stability. Such a high stability could be attributed to the favorable microenvironment that maintains the GOD activity and prevents the leakage of enzyme.The practical applications of the designed biosensor were evaluated by the determination of glucose recovery in human blood serum. The recovery was investigated by spiking with different concentrations of glucose to serum sample. The samples were diluted 1,000 times before determination. The analytical results are shown in Table In this work, a novel electrochemical GOD biosensor based on PtAuNP/ss-DNA/GR nanocomposites was developed for the determination of glucose. The bionanocomposite film provided a suitable microenvironment, which could effectively present a large loading amount of enzyme and enhanced the direct electron transfer between the enzyme's active sites and the electrode. The modified electrode exhibited excellent analytical performance with wide linear range, low detection limit, and good selectivity for measuring glucose. Therefore, the composite of PtAuNPs/ss-DNA/GR is a good material platform, promising for construction of the third-generation enzyme biosensor, biofuel cells, and bioelectrochemical devices.EDS: energy-dispersive X-ray spectrometer; GOD: glucose oxidase; GR: graphene; PtAuNPs: PtAu bimetallic nanoparticles; ss-DNA: single-stranded DNA; TEM: transmission electron microscopy.The authors declare that they have no competing interests.The manuscript was written through the contributions of all authors, JL, W-MW, L-ML, LB, and X-LQ. All authors read and approved the final manuscript.JL is an undergraduate student at Jiangxi Agricultural University. W-MW, L-ML, LB, and X-LQ are teachers at Jiangxi Agricultural University.


Anopheles stephensi mitochondrial malic enzyme (ME) emerged as having a relevant role in the provision of pyruvate for the Krebs' cycle because inhibition of this enzyme results in the complete abrogation of oxygen uptake by mitochondria. Therefore, the identification of ME in mitochondria from immortalized A. stephensi (ASE) cells and the investigation of the stereoselectivity of malate analogues are relevant in understanding the physiological role of ME in cells of this important malaria parasite vector and its potential as a possible novel target for insecticide development.To characterize the mitochondrial ME from immortalized ASE cells , mass spectrometry analyses of trypsin fragments of ME, genomic sequence analysis and biochemical assays were performed to identify the enzyme and evaluate its activity in terms of cofactor dependency and inhibitor preference.Anopheles gambiae (98%) and 59% homologous to the mitochondrial NADP+-dependent ME isoform from Homo sapiens. Measurements of ME activity in mosquito mitochondria isolated from ASE cells showed that (i) max Vwith NAD+ was 3-fold higher than that with NADP+, (ii) addition of Mg2+ or Mn2+ increased the max Vby 9- to 21-fold, with Mn2+ 2.3-fold more effective than Mg2+, (iii) succinate and fumarate increased the activity by 2- and 5-fold, respectively, at sub-saturating concentrations of malate, (iv) among the analogs of L-malate tested as inhibitors of the NAD+-dependent ME catalyzed reaction, small (2- to 3-carbons) organic diacids carrying a 2-hydroxyl/keto group behaved as the most potent inhibitors of ME activity .The encoding gene sequence and primary sequences of several peptides from mitochondrial ME were found to be highly homologous to the mitochondrial ME from Anopheles stephensi ME is of critical relevance given its important role in bioenergetics, suggesting that it is a suitable target for insecticide development.The biochemical characterization of  Anopheles stephensi [Anopheles species in the investigation of malaria transmission [Aedes aegypti [A. stephensi Mos. 43 cell line)] from A. stephensi, as well as flight muscle mitochondria of a beetle (Popillia japonica), which also have the ability to oxidize proline at a high rate, have been shown to contain an unusually active malic enzyme [+ preferentially as a coenzyme and presumably produces pyruvate by the oxidative decarboxylation of malate [Recently, several pathways for energy production have been identified in mitochondria from tephensi , a well-smission . The mitsmission , the mos aegypti  as well  aegypti . About 2 aegypti , whereas aegypti , but thi aegypti , consist aegypti . Mitochoc enzyme . The latf malate . This miL-malate to pyruvate and CO2 with the concomitant reduction of the cofactor NAD+ or NADP+ [2+, Mn2+, or others) in the catalysis of this reaction. ME activity was first isolated from pigeon liver [+-dependent ME , but prefers NAD+ under physiological conditions. In invertebrates, and in particular in insects, unusually high activity of NAD+-linked malic enzyme has been reported in flight muscle mitochondria of the beetle Popillia japonica [Malic enzyme  catalyses the reversible oxidative decarboxylation of or NADP+ . The enzon liver  and has on liver  and biosE , mitochE , and miE , which japonica  and fromjaponica .Based on previous reports , ASE mitOrganic acids were purchased from Sigma Chemical Co. . All reagents were of analytical grade.A. stephensi ASE cell line was grown in modified Eagle's minimal essential medium ("E5") supplemented with glucose, L-glutamine, vitamin solution, nonessential amino acids, penicillin and streptomycin, and 5% heat-inactivated fetal bovine serum at 28°C with 5% CO2 as described [The immortalized escribed . The pop6 cells. Protein concentration was determined by using the BCA Protein Assay (Pierce).Cells were centrifuged for 1 min at 500 g at 4°C and mitochondria were isolated from pelleted cells as described [+, 50 mM HEPES (pH 7.8), and 3 mM of MnCl2 (unless indicated otherwise) were added. The reaction was initiated with the addition of 40 μg of mosquito mitochondrial lysate protein. The change in absorbance was measured using the Cary 1E Spectrophotometer at 340 nm for 2-3 minutes (control). The ME specific activity was calculated for each trace utilizing the extinction coefficient for NADH at 340 nm (6.22 mM-1 cm-1). Each L-malate analogue was added to the reaction mixture at the indicated concentrations and the change in absorbance was measured for another 3-5 minutes.The ME enzymatic assay was performed using a method outlined by  with theA. gambiae protein database  assuming the digestion enzyme trypsin. X! Tandem was searched with a fragment ion mass tolerance of 0.40 Da and a parent ion tolerance of 1.8 Da. Iodoacetamide derivative of cysteine was specified in X! Tandem as a fixed modification. Deamidation of Asn and Gln, oxidation of Met and Trp, sulphone of Met, Trp oxidation to formylkynurenin of Trp and acetylation of the N-terminus were specified in X! Tandem as variable modifications. Scaffold (version Scaffold-3_00_08) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 90.0% probability as specified by the Peptide Prophet algorithm [LC-MS/MS analyses were performed at the Proteomics Facility of the University of California Davis Genome Center. Tandem mass spectra were extracted by BioWorks version 3.3. Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analysed using X! Tandem ,19. X! Tlgorithm . Proteinlgorithm . ProteinA. stephensi ME, the A. gambiae ME amino acid sequence (Q7QB64) was used as a query by Dr. Zhijian (Jake) Tu (Virginia Tech) to identify homologous sequence in the June 2010 unpublished draft of the A. stephensi assembly by TBLASTN . The region with the best match plus 1-kb flanking sequences on either side were retrieved.To identify the full-length coding sequence for t-test  with p ≤ 0.05 considered as statistically significant.The experiments were run in duplicate or triplicate and repeated two times in independent experiments. Data were expressed as mean ± SEM. The data were evaluated by using the A. stephensi cells, Multidimensional Protein Identification Technology (MudPIT) was used. This technique involves digesting mosquito proteins with trypsin and separating the resulting peptides with two liquid column chromatography steps: the first being a strong cationic exchange, and the second being reversed-phase HPLC. As the peptides elute from the second column, they are sprayed into a linear ion trap mass spectrometer. The first MS scan assigned each peptide a mass/charge ratio. The most intense peptide signals are then fragmented in a second MS/MS scan, which assigns each peptide a unique "fingerprint" , flies , ants , lice (Pediculus spp. 73%), moth (Bombyx spp. 64%), and a nematode (Ascaris suum 54%). BLAST query against the human database revealed a 59% identity to the human mitochondrial NADP+-dependent ME (or ME-3), followed by a 57% and a 55% identity to the human cytosolic ME-1 and the human mitochondrial ME-2, respectively. The lower homology of the A. stepephensi ME with the human mitochondrial counterpart was not due to the mitochondrial targeting sequence because its removal from the human isoform and realignment of the mature protein with the mosquito protein did not change the identity (59%), similarity (75%) or score (1742). Of note, the two human mitochondrial ME isoforms share only 54% of their amino acids, which is the same range of homology when comparing human MEs with A. stephensi ME (this study) or from maize chloroplasts (47%). By comparison the highly homologous A. gambiae ME (Q7QB64) is 59% identical to the human ME-3, 57% to human ME-1, and 55% to human ME-2, whereas the other ME from A. gambiae (Q7PRY4) has 60% identity to the human ME-1, 58% to ME-3, and 53% to ME-2. As in A. gambiae, two MEs have been identified for A. darlingi, in which one of them (E3WUS4) is 62% identical to the human ME-1, while the other ME (E3WW73) is 60% identical to the human ME-3. Thus, for these mosquito species, no ME-2 ortholog  is apparent. In agreement with these findings, only one mitochondrial ME from A. stephensi was detected in the performed MudPIT analyses and genome sequence analyses. However, a possible A. stephensi ME paralog with approximately 60% identity to A. gambiae ME (Q7QB64) was detected . While this encoded protein is potentially interesting, we have focused on the highly homologous A. stephensi ME here.BLAST query of the complete amino acid sequence of A. stephensi ME, a high-quality prediction of the 3D structure and biological function of the ME was performed by using a template-based modelling platform [A. stephensi ME (with a C-score of 1.985) was closely related to that of Ascaris suum , human ME-2 (1pj3A and 1gz4A) and Ascaris suum , but the addition of either metal increased significantly the maximum activity (9 to 21-fold); however, Mn2+ was a better cofactor than Mg2+  and 35 U/mg protein, respectively, whereas the activity of the human cytosolic NADP+-ME is 40 to 55 U/mg [+ or NADP+; however, max Vwith NAD+ was 3-times higher than that with NADP+ (Table vide supra) and structure (vide infra).To gain insight in the activity of ME, enzymatic parameters were evaluated in mosquito mitochondria isolated from ASE cells. The production of NAD(P)H was followed in the presence of malate and NAD(P) 55 U/mg . ASE ME Ascaris suum ME has an Asp, suggesting that these isoforms, like the human ME-2, may be activated by fumarate by conserving the negative charge at this position. On the other hand, in the mosquito ME, Leu occupies the equivalent position of Arg-67 , suggesting that mosquito ME should not be activated by fumarate. In addition to Arg-67, it has been reported that in the nematode mitochondrial ME, the residues that are involved in the binding of fumarate are Ala-78, Leu-81 and Leu-105 . Therefore the presence or absence of this Cys or Ser residue in this binding fold apparently distinguishes the preference for NADP+ over NAD+ as suggested before by others [The proposed ADP binding beta-alpha-beta fold is shown which contains a characteristic arrangement of Gly residues and nonpolar and hydrophilic amino acids at specific positions  because it is believed that disrupting the close packing of the secondary structure allows the structure to accommodate the phosphate moiety of NADP+ [+ binding preference over that of NADP+ [4 NADP+-ME [4 NADP+-ME showing that residues at both sites are part of the NADP+-binding site justifying the high degree of conservation among all NAD(P)+-ME [The fingerprint region of NAD(P)tructure ,31. Thisof NADP+ . Furtherof NADP+ . HoweverAla-X-X-X-Ala; Figure +-binding domains relative to NAD+-binding domains, whereas Gly residues were more consistently identified in NAD+-dependent enzymes [A. stephensi ME have an Ala-X-X-X-Ala segment, regardless of whether they use NADP+ or NAD+.It has been suggested that two Ala residues spaced by three amino acids , nematode ME, human ME-2 and bacteria ME, this sequence position corresponds to a Lys, His, Gln and Glu, respectively , this Lys is replaced by Ser  is ME-1 . Additio is ME-1 . Sequencr Figure  arrow. I2 Figure  asterisk2 Figure  arrow ar+ and malate with i Kvalues from 80 to 430 μM [et al [Ascaris ME [+ or L-malate in A. stephensi mitochondrial ME at the concentrations tested . The Arg-197, Arg-542 and Arg-556 residues in human ME-2 appear to be involved in the binding of ATP at the exosite [ATP acts as an active-site inhibitor of mitochondrial ME-2 following a competitive mechanism for NADo 430 μM ,35. AccoM [et al , the precaris ME ,38. In a exosite . These r+-dependent ME catalyzed reaction  to 0.6 μM  are consistent with this mechanism, for these compounds are either intermediates  or structurally similar intermediates  that fit the tight active site of the enzyme [The catalysis by ME generally proceeds in two steps, namely the dehydrogenation of malate to produce oxaloacetate, and then the decarboxylation to produce pyruvate. In a proposed enzymatic catalysis based on structural data the crystallography data, the latter step is believed to occur through the formation of a keto-enol intermediate structurally similar to oxalate . The stre enzyme . Besidese enzyme . In supp+ and/or hydrogen bonding with the side chain amide of Asn and the 2'-hydroxyl of the nicotinamide ribose [Flaveria trinervia, the presence of a group with a low pK value (6 to 6.6), probably an H residue, was responsible for the binding of the 2-hydroxyl of malate and transfer of the hydride to form the intermediate oxaloacetate [Within the diacids, the presence of a keto group in position 2 seemed to be an important factor in controlling the inhibitory effect of the compounds studied, for lack of this moiety in, for example, succinate or fumarate, resulted in a loss of the inhibitory effect. The change of the 2-keto group of the alpha-ketobutyric acid to 2-hydroxy  or the transfer to the 3-hydroxy (beta-hydroxybutyrate) resulted in negligible effect on the ME activity Figure . Thus, te ribose . These roacetate . These rCaenorhabditis elegans, Arabidopsis thaliana, and rice. The mammalian ME-3 isoform has a strong tissue-specific expression, mostly in organs with a low division rate  or withd brain; . It has le cells . Thus, tA previous report indicated that addition of tartronic acid completely inhibited the oxygen consumption of Pro-supplemented phosphorylating mitochondria . This su+ over NADP+, is to provide NADPH for the detoxification of reactive oxygen radicals generated in mitochondria during respiration. Glutathione reductase activity requires NADPH to recycle oxidized glutathione for the glutathione peroxidase system [+-ME might contribute to the generation of reducing equivalents.It is likely that a secondary role of this enzyme in mosquito mitochondria, based on the preferential use of NADe system , and mitThe critical role of ME in the bioenergetics of mosquitoes along with the deeper understanding of its biochemistry and chemical requirements for inhibition, suggests that this enzyme could became a suitable target for insecticide development. In this regard, structural analogues of L-malate  behaved as the most potent inhibitors of ME activity .A. gambiae demonstrated that exposure to permethrin induced strong upregulation of genes that encode subunits of Complex I [Schizosaccharomyces pombe to rotenone, a potent Complex I inhibitor [Enzymes significantly associated with insecticide resistance include esterases and cytochrome P450s involved in the oxidative catabolism of insecticides . Mitochonhibitor . In addinhibitor ,52. Thesnhibitor ,54.Anopheles stephensi Mos. 43 cell line; ME: malic enzyme; ME-1: cytosolic NADP+-dependent ME; ME-2: mitochondrial NAD(P)+-dependent ME; ME-3: mitochondrial NADP+-dependent ME; MS: mass spectrometry; MS/MS: tandem MS; mtDNA: mitochondrial DNA; MudPIT: Multidimensional Protein Identification TechnologyASE: Drs. Giulivi and Luckhart filed a disclosure and record of invention with the University of California Davis on 04/11/2008 entitled "Inhibitors of mosquito malic enzyme as insecticides" (UC Case #2008-673-1).JP carried out all enzymatic analyses and performed statistical analyses. EN supervised directly the technical work, performed statistical analyses and helped to draft the manuscript. SL provided all biological materials, contributed with substantial intellectual input in regards to the malaria field, and revised the manuscript critically. CG conceived of the study, participated in its design and coordination, performed all sequence alignment and analyses, protein modelling and interpretation, and wrote the manuscript. All authors read and approved the final manuscript.A. stephensi MEDNA and protein sequences for . A. Nucleotide sequence for A. stephensi ME. B. Amino acid sequence deduced from DNA equence. C. Protein sequence producing significant alignment to A. stephensi ME performed with BLAST.Click here for fileA. stephensi MEModeling results obtained with . This file contains the results obtained with the simulation suite named I-TASSER performed with the primary amino acid sequence of A. stephensi ME.Click here for fileAdditional information on the nomenclature of proteins listed under Figure 4. This file contains all additional information  in regards to the nomenclature of ME shown under Figure 4.Click here for file